Mitochondrial (mt) DNA from Paramecium aurelia is a linear molecule replicating via a lariat (circle plus tail) intermediate and terminating in a linear head-to-head dimer length molecule. The nature of the head-to-head dimer molecule has allowed us to clone restriction fragments of this region and commence sequence analysis. The results thus far indicate that for both species 1 and 4, the center of this head-to-head initiation region consists of an At-rich direct repeat which is non-palindromic. Flanking this region for each species is a palindromic region. Now we plan to clone the monomer fragment to determine whether these non-palindromic AT repeats are present to the same degree or whether some processing has occurred on one or the other strand. This would have some bearing on the mechanism of replication for this linear molecule. Using these cloned initiation regions we will also isolate and characterize those proteins which bind to DNA affinity columns. In addition, we have constructed detailed restriction endonuclease maps for species 1 and 4 and shown that each have the same general structure. Both have one copy of each rRNA gene and these genes are far apart on the genome. The large ribosomal RNA subunit is located near the termination end of the molecule with the small RNA subunit being more centrally located. Clones for these genes from both species 1 and 4 have been constructed and we plan to sequence these genes in order to determine sequence diversity and gene expression regulation.